ABSTRACT
In this study, biomarkers and transcriptional factor motifs were identified in order to investigate the etiology and phenotypic severity of Down syndrome. GSE 1281, GSE 1611, and GSE 5390 were downloaded from the gene expression ominibus (GEO). A robust multiarray analysis (RMA) algorithm was applied to detect differentially expressed genes (DEGs). In order to screen for biological pathways and to interrogate the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, the database for annotation, visualization, and integrated discovery (DAVID) was used to carry out a gene ontology (GO) function enrichment for DEGs. Finally, a transcriptional regulatory network was constructed, and a hypergeometric distribution test was applied to select for significantly enriched transcriptional factor motifs. CBR1, DYRK1A, HMGN1, ITSN1, RCAN1, SON, TMEM50B, and TTC3 were each up-regulated two-fold in Down syndrome samples compared to normal samples; of these, SON and TTC3 were newly reported. CBR1, DYRK1A, HMGN1, ITSN1, RCAN1, SON, TMEM50B, and TTC3 were located on human chromosome 21 (mouse chromosome 16). The DEGs were significantly enriched in macromolecular complex subunit organization and focal adhesion pathways. Eleven significantly enriched transcription factor motifs (PAX5, EGR1, XBP1, SREBP1, OLF1, MZF1, NFY, NFKAPPAB, MYCMAX, NFE2, and RP58) were identified. The DEGs and transcription factor motifs identified in our study provide biomarkers for the understanding of Down syndrome pathogenesis and progression.
Subject(s)
Animals , Humans , Mice , Rats , Amino Acid Motifs/genetics , Computational Biology/methods , Down Syndrome/genetics , Gene Regulatory Networks/genetics , Transcription Factors/analysis , Algorithms , Biomarkers/analysis , Databases, Genetic , Down Syndrome/etiology , Gene Expression , Gene Ontology , Molecular Sequence Annotation/methods , Phenotype , Protein Array Analysis/methods , Up-Regulation/geneticsABSTRACT
The objectives of this study were to standardize a PCR-RFLP genotyping method for the AY_731081:g.1900T>C SNP of the equine CD14 gene, and to characterize this SNP and two other polymorphisms (AY_005808: c.1530A>G of the TLR4 gene and AX_463789: g.133T>C of the Cε gene) in Mangalarga horses, in order to contribute to future studies investigating the association between DNA markers and traits related to immune system physiology in this breed. A total of 151 Mangalarga horses of both sexes and variable ages, representative of the population of São Paulo State, were used. PCR-RFLP was found to be adequate for genotyping of the AY_731081: g.1900T>C SNP of the equine CD14 gene. However, this polymorphism is probably not present in Mangalarga horses, thus impairing association studies using this marker in the breed. The population genetic parameters obtained for the TLR4 AY_005808:c.1530A>G and Cε AX_463789:g.133T>C polymorphisms suggest the use of these markers in association studies with immune system-related traits in Mangalarga horses.
Os objetivos deste trabalho foram a padronização da metodologia PCR-RFLP para genotipagem do SNP AY_731081:g.1900T>C do gene CD14 equino, bem como a caracterização em equinos da raça brasileira Mangalarga deste e de outros dois polimorfismos, o AY_005808: c.1530A>G do TLR4 e o AX_463789: g.133T>C do Cε, a fim de promover o embasamento necessário para futuras pesquisas visando à associação entre marcadores de DNA e características relacionadas à fisiologia do sistema imune na raça. Para tanto, foram utilizados 151 animais Mangalarga, de ambos os sexos e de idades variadas, representativos da população do estado de São Paulo. O método de PCR-RFLP mostrou-se adequado para a genotipagem do SNP AY_731081: g.1900T>C do gene CD14 equino. Entretanto, tal polimorfismo provavelmente não ocorre em equinos Mangalarga, impossibilitando estudos de associação com o marcador na raça. Os parâmetros genético-populacionais obtidos para os polimorfismos AY_005808:c.1530A>G do gene TLR4 e o AX_463789:g.133T>C do gene Cε demonstraram a possibilidade de realização de pesquisas.
Subject(s)
Animals , Horses/genetics , Polymorphism, Genetic , Genotyping Techniques/veterinary , Molecular Sequence Annotation/methods , Polymerase Chain Reaction/veterinaryABSTRACT
Introducción. El estudio del ácido desoxirribunocleico (DNA) es imprescindible para la medicina moderna; por ello, se ha diseñado diferentes técnicas para su extracción, cuyos costos son altos y de tiempo prolongado. En este trabajo se describe un método modificado de extracción de DNA (DNA-UMSAgen) basado en la técnica de Miller. Métodos. Las células mononucleares fueron obtenidas de sangre venosa periférica de un sujeto voluntario, para realizar 40 extracciones de DNA; 20 con el método modificado DNA_UMSAgen y otros 20 con el método clásico. Posteriormente se evaluó la concentración, calidad y utilidad de estos DNA extraidos. Resultados. La pureza del DNA extraido por el método DNA-UMSAgen es de 1,88 similar al de la técnica clásica 1,91, las concentraciones obtenidas son 20,4 ug/106 cel y 56ug/106 cel respectivamente. La evaluación por electroforesis en agarosa y la amplificación del exon 12 del gen JAK2 por PCR fue satisfactoria. Conclusión. El método DNA-UMSAgen es una alternativa de extracción de DNA genómico, rápido y económico, adecuado para paises en vias de desarrollo.
INTRODUCTIONDNA is the genetic material of the cell. Actually for its study, laboratory techniques are available that require its extraction free of impurities. This paper describes the DNA-UMSAgen method as an alternative for DNA extraction which is based on the Miller technique.METHODSThe mononuclear cells were obtained of venous peripheral blood of a voluntary subject, for to realize 40 DNA's extractions; 20 with the modified method DNA-UMSAgen and other 20 with the classic method. Later there was evaluated the concentration, quality and utility of these extracted DNA.RESULTSThe DNA purity extracted by the DNA-UMSAgen method is 1,88 similar to the classic technique 1,91, the concentration obtained were 20,4 ug/106 cells and 56ug/106 cells respectively. The evaluation by agarose gel electrophoresis and the amplification of the exon 12 of the JAK2 gen by PCR was successful.CONCLUSIONThe DNA-UMSAgen extraction method is a very acceptable fast, easy and inexpensive alternative method for underdeveloped countries for DNA extraction.